PMC

The distributions of the lysines (K) and arginines (R) are shown in pink and red respectively.

Secondary plot of the reciprocal of V_max against inhibitor concentration ⧫ RgpB and ▪ rKgp. The K_i’ values were obtained from the x-intercept.

Assays were performed using the chromogenic substrates GPKNA (□) and BapNA (○). The % proteolytic activity was also determined using the fluorescent substrate DQ-BSA with rKgp (▪) and RgpB (•).

(A) Models of Kgp and RgpB based on the coordinates from PDB (1cvr.pdb), highlighting the cysteine residues. (B) Model of RgpB highlighting the His-Cys Cα distances in the two caspase sub-domains.

(A) Model of RgpB highlighting the N-terminus, catalytic Cys and His residues, and residues that differ between strains in red. The residues that form a surface-exposed conserved patch are predicted to interact with the propeptide. (B) Schematic representation of the inhibition of Kgp by its propeptide. Kgp was modelled using Orchestrar from within Sybyl-8.1 and based on the X-ray crystal structure of RgpB 1cvr.pdb . The propeptide is based on the A chain of the X-ray crystal structure of RgpB interacting with its propeptide 4ief.pdb .

(A) Time course of rKgp measured as change in absorbance (405 nm) without an inhibitor (•) and with 40 mg/L Kgp propeptide (Kgp-PP) with (□) and without the hexahistidine tag (▵) at 1 mM cysteine in the assay with the Lys-specific chromogenic substrate (GPKNA). The final concentration of rKgp per well is 1.16 mg/L. (B) Time course of RgpB using the fluorescent natural substrate DQ-BSA without RgpB-PP (▪), with 10 mg/L RgpB-PP (⋄) and 1 mM TLCK (□).

(A) Non-reducing SDS-PAGE of recombinant Kgp propeptide expressed in E. coli. Lane 1: See-Blue® Pre-stained standard, where sizes in kDa are indicated; Lane 2: Flow through the Ni Column; Lane 3: Wash from Ni column; Lane 4: Thrombin cleaved product; Lane 5: Total thrombin-free Kgp propeptide extract prior to size exclusion chromatography. The gel was stained with Coomassie® Brilliant Blue (G250). (B) Size exclusion chromatogram (Superdex G75) of Kgp propeptide revealing dimerisation. (C) SDS-PAGE of the purified Kgp-propeptide monomer and dimer forms incubated with and without 5 mM DTT but without boiling revealing the disruption of the dimer with 5 mM DTT.

(A) Reduced SDS-PAGE analysis of incompletely processed precursors observed in Day 1 and 3 culture supernatants of P. gingivalis ECR368 that releases rKgp. (B) SDS-PAGE analysis of the ∼60 kDa precursor form of rKgp with and without DTT revealing a single band under reducing conditions and two bands under non-reducing conditions, highlighting the disulphide bridge (CYS–CYS) that forms between the N-terminal half of the propeptide and the mature protease as summarized in (C) Processing steps: The N-terminal half of the propeptide is represented by the white rectangle, the C-terminal half by the black rectangle and the mature Kgp by the hatched rectangle.